With an incidence of approximately one in 750 live births, Down syndrome (DS) is the most common genetic cause of intellectual disability (ID). Although ID can be mild, the average IQ is ~40-50. In the United States, the population of people with DS is currently estimated at ~350,000 and continues to increase as the average life expectancy increases. Pharmacotherapies for cognitive deficits in DS would, therefore, have a significant impact. Currently, there is enthusiasm for initiating clinical trials for ID in DS base on rescue of learning/memory deficits in one mouse model of DS, the Ts65Dn. However, three factors may limit success. First, the Ts65Dn is trisomic for only 88 of 161 human chromosome 21 (HSA21) protein genes; non-trisomic HSA21 orthologs include some that cause L/M deficits and impaired synaptic plasticity when they are over expressed. Over expression of these genes in DS will affect the molecular basis of the phenotype and, possibly, drug responses. Second, genetic heterogeneity in the human population may also alter phenotype and drug responses. Third, a single drug may be insufficient to correct the many molecular and cellular perturbations that contribute to the complexity of human cognitive deficits. To address these three issues, we propose to use cultured human primary neurons and test combinations of drugs. Relative to controls, neuronal cultures from DS fetal brain show decreased synapse density, decreased mitochondrial function, and increased levels of reactive oxygen species. In Aim 1, we will test DS fetal brain neuronal cultures for rescue of these abnormalities by separately treating with eight different drugs, each of which has been shown or is predicted to be effective in the Ts65Dn or DS. In Aim 2, we will use a novel experimental/computational method, Feedback System Control (FSC), to test combinations of the same drugs for rescue of each DS abnormality. Currently, identifying drug/dose combinations to treat disease is based on high-throughput random drug screenings or trial-and-error testing in a clinical setting. In contrast, FSC provides a systematic search paradigm that combines experimental testing, in a cell system, with use of a computer algorithm to find biologically effective drug/dose combinations. Critically, FSC has shown that biological responses are smooth over a wide range of drugs/doses. Therefore the search rapidly converges to an optimal readout, requiring testing of only 200-300 drug/dose combinations, not hundreds of thousands. In Aim 3, we will extend FSC to Cascade FSC to optimize the drug combination for simultaneous rescue of multiple DS abnormalities. The important advantages of the system we propose are that i) it uses a complete human trisomy HSA21 neuronal model, ii) as a cell system, it allows rapid screening of many drugs, iii) a combination of drugs is more likely to be effective in rescuing multiple pathway abnormalities and therefore in rescuing circuits underlying multiple, complex cognitive failures, iv) the concentrations required for each drug in the combination likely will be lower tha those required when any drug is used alone; this will decrease potential negative interactions and off target effects; and v) only 200-300 drug/dose combinations need to be tested, not hundreds of thousands. The goal of this application is to identify the optimal doses of a combination of drugs that together rescue multiple abnormalities present in DS neurons. In future studies, the final converged drug combination will be tested in cortical neuronal cultures for rescue of additional abnormalities, in DS-derived neurally differentiated induced pluripotent stem cells (iPSCs), and, as a final preclinical trial evaluation, in a complete DS mouse model for rescue of L/M deficits. This effort is a novel approach to increase the probability that clinical trials for cognitive deficits in DS will be both safe and effective.